title: Identification and characterization of an anti-fungi Fusarium oxysporum f. sp. cucumerium protease from the Bacillus subtilis strain N7.
abstract: A newly discovered alkaline antifungal protease named P6 from Bacillus subtilis N7 was purified and partially characterized. B. subtilis N7 culture filtrates were purified by 30-60% (NH4)2SO4 precipitation, anion-exchange chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band of 41.38 kDa. Peptide sequence of protease P6 was determined using a 4800 Plus MALDI TOF/TOF™ Analyzer System. Self-Formed Adaptor PCR (SEFA-PCR) was used to amplify the 1,149 bp open read frame of P6. Dimensional structure prediction using Automatic Modeling Mode software showed that the protease P6 consisted of two β-barrel domains. Purified P6 strongly inhibited spore and mycelium growth of Fusarium oxysporum f. sp. cucumerium (FOC) by causing hypha lysis when the concentration was 25 μg/ml. Characterization of the purified protease indicated that it had substrate specificity for gelatin and was highly active at pH 8.0-10.6 and 70°C. The P6 protease was inhibited by EDTA (2 mmol/L), phenyl methyl sulfonyl fluoride (PMSF, 1 mmol/L), Na(+), Fe(3+), Cu(2+), Mg(2+) (5 mmol/L each) and H2O2 (2%, v/v). However, protease activity was activated by Ca(2+), K(+), Mn(2+) (5 mmol/L each), mercaptoethanol (2%, v/v) and Tween 80 (1%, v/v). In addition, activity was also affected by organic solvents such as acetone, normal butanol and ethanol, but not hexane (25%, v/v each).
authors.affiliation: Jiangsu Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Luo
authors.forename: Lifei
authors.initials: L
authors.lastname: Sun
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhu
authors.forename: Wei
authors.initials: W
authors.lastname: Ran
authors.forename: Qirong
authors.initials: Q
authors.lastname: Shen
mesh_terms: D000935:Antifungal Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005670:Fusarium / Q000187:drug effects*; D016366:Open Reading Frames / Q000235:genetics; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000935:Antifungal Agents; D010447:Peptide Hydrolases